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Biomaterials 112 (2017) 275e286

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Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

Click chemistry improved wet adhesion strength of mussel-inspiredcitrate-based antimicrobial bioadhesives

Jinshan Guo a, 1, Gloria B. Kim a, 1, Dingying Shan a, Jimin P. Kim a, Jianqing Hu b,Wei Wang c, Fawzi G. Hamad d, Guoying Qian c, Elias B. Rizk e, Jian Yang a, *

a Department of Biomedical Engineering, Materials Research Institute, The Huck Institutes of the Life Sciences, The Pennsylvania State University, UniversityPark, PA, 16802, USAb School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou, 510640, Chinac Zhejiang Provincial Top Key Discipline of Bioengineering, College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo, 315100,Chinad Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA, 16802, USAe Department of Neurosurgery, College of Medicine, The Pennsylvania State University, Hershey, 17033, USA

a r t i c l e i n f o

Article history:Received 7 August 2016Received in revised form3 October 2016Accepted 8 October 2016Available online 12 October 2016

Keywords:Click chemistryBioadhesivesMusselCitric acidAntimicrobial

* Corresponding author. W340 Millennium Science16802, USA.

E-mail address: jxy30@psu.edu (J. Yang).1 These authors contribute equally to this work.

http://dx.doi.org/10.1016/j.biomaterials.2016.10.0100142-9612/© 2016 Elsevier Ltd. All rights reserved.

a b s t r a c t

For the first time, a convenient copper-catalyzed azide-alkyne cycloaddition (CuAAC, click chemistry)was successfully introduced into injectable citrate-based mussel-inspired bioadhesives (iCMBAs, iCs) toimprove both cohesive and wet adhesive strengths and elongate the degradation time, providingnumerous advantages in surgical applications. The major challenge in developing such adhesives was themutual inhibition effect between the oxidant used for crosslinking catechol groups and the Cu(II)reductant used for CuAAC, which was successfully minimized by adding a biocompatible buffering agenttypically used in cell culture, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), as a copperchelating agent. Among the investigated formulations, the highest adhesion strength achieved(223.11 ± 15.94 kPa) was around 13 times higher than that of a commercially available fibrin glue(15.4 ± 2.8 kPa). In addition, dual-crosslinked (i.e. click crosslinking and mussel-inspired crosslinking)iCMBAs still preserved considerable antibacterial and antifungal capabilities that are beneficial for thebioadhesives used as hemostatic adhesives or sealants for wound management.

© 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Approximately 114 million surgical and procedure-basedwounds occur every year worldwide and it is expected that theglobal wound closure market would reach $14 billion by 2018 [1].Currently, approximately 60% of wound closure procedures are stillperformed with sutures, staples, and other mechanical methods[2]. However, these methods often require special surgical and/ordelivery tools and follow-up visits are needed if nondegradablesutures are used, and sometimes liquid or air leakage is inevitable.Therefore, bioadhesives have obtained much attention as a cost-effective technology for wound closure. In the past three decades,bioadhesives have transformed clinical surgical practices in both

Complex, University Park, PA,

external and internal lacerations by promoting tissue healing whilepreventing blood loss [3e7]. However, most current adhesives (i.e.a biologically-derived fibrin glue [4,7,8]) cannot provide sufficientmechanical support especially when used in wet environmentsduring surgical procedures. Inspired by the adhesion strategy ofmarine mussels, a new family of biomimetic, mussel-inspired ad-hesives has become an area of intense research. Mussel-inspiredpolymers are synthesized from catechol-containing amino acidssuch as L-3,4-dihydroxy-phenylalanine (L-DOPA), typically derivedfrom various mussel adhesion proteins, known to contribute to thestrong wet adhesion strength of marine mussels to non-specificsurfaces [5,9e13]. Among those polymers, injectable, citrate-based, mussel-inspired bioadhesives (iCs) [11] and antimicrobialiCs [12] developed in our group have been acknowledged for theircost-effective and convenient syntheses along with vastlyimproved wet adhesion strength as compared to that of fibrin glue.

However, mussel-inspired polymers including iCs commonlysuffer from insufficient cohesive strength under wet conditions, as

J. Guo et al. / Biomaterials 112 (2017) 275e286276

polymers can easily detach from adhered surfaces by deformationor stretching [10e13]. Strong wet mechanical strength is particu-larly vital for in vivo applications, hence our goal was to furtherchemically modify iCs to optimize this property. The catecholgroups in previously reported formulations of iCs participate in theformation of crosslinked polymer networks, reducing the numberof available catechol groups that can chemically react with wettissues [10e13]. Moreover, the rapid degradation rate of iCs re-mains a significant challenge as it may lead to undesirable earlystructural collapse and failure of wound closure. In our previousstudy, click chemistry was introduced into a citrate-based, poly-ester elastomer to yield a mechanically robust, surface-clickable,and biodegradable poly (1, 8-octanediol citrate) (POC-click)[14e18]. Propelled by the successful incorporation of click chem-istry into POC-click materials, click chemistry was introduced inorder to synthesize more mechanically robust iCs. The rationalebehind modifying iCs with click chemistry are as follows: 1) thetriazole rings formed by click chemistry could imitate amide bondsand serve as cohesive strength-improving moieties in polyester-based iCs; 2) click crosslinking could spare more catechol groupsfrom network formation to participate in adhesion, thus furtherimproving adhesion strength; 3) to slow down the initial degra-dation rate of iCs and sustain mechanical integrity in the earlytissue regeneration stage by using click chemistry; 4) to achievefirst-slow-then-fast degradation without prolonging total degra-dation time via click modification (similar to POC-click) [14]; and 5)to enable convenient bioconjugation after or concurrent withcrosslinking.

One major concern in applying click chemistry for biomedicalapplications is that the copper catalysts often used in click reactionscan also catalyze the production of reactive oxygen species (ROS)that are harmful to cells [19]. In addition, copper-free thermal clickreactions used to produce the aforementioned POC-click materialstypically occur at 100 �C so they are not suitable for crosslinkingbioadhesive polymers due to the low use temperature of bio-adhesives, usually around the body temperature of 37 �C. Analternative to thermal click chemistry is strain-promoted azide-alkyne cycloaddition (SPAAC), another copper-free click chemistrythat can be used for forming hydrogels at room or body tempera-ture [20e22], though strain-promoted triple-bond containing re-agents are not readily available for this method. With its highreaction rates and nearly quantitative yields, CuAAC is still widelyused for synthesizing hydrogel-based biomaterials and encapsu-lating cells [23e29]. To reduce the cytotoxicity of copper catalystsystems, water-soluble copper-chelating ligands/agents have beenused, as they include copper-chelating azides [19], bis(L-histidine)[30], tris-(hydroxypropyltriazolylmethyl)amine [31], bipyridine[32], and bis[(tert-butyltriazoyl)methyl]-[(2-carboxymethyltriazoyl)-methyl]-amine (BTTAA) [33]. All of theseligands accelerate the CuAAC reaction and act as sacrificial re-ductants, helping to protect cells and bioactive molecules from thedamage caused by ROS produced from the Cu-catalyzed reductionof oxygen [19,34]. However, the toxicity and lowwater-solubility ofsome of these copper-chelating ligands/agents still remainproblematic.

Here, we present a dual crosslinking iC system as a new strategyto address the challenges related to the low cohesive strength andrapid degradation of existing mussel-inspired adhesives. In detail,azide- or alkyne-functionalized iCs were synthesized by addingpentaerythritol triazide (3N3) or trimethylolethane dipropiolate(2PL) monomers to the side groups of iCs to produce iC-3N3 or iC-2PL, respectively (Scheme 1). Alkyne-functionalized gelatin(Gelatin-Al, GL) (Scheme S1) was also synthesized and introducedinto the iC-X (X ¼ click) system to further improve the mechanicalstrength and biocompatibility of the system. The crosslinking of iC-

X system can be initiated by the use of either an oxidant (ie. sodium(meta) periodate (PI) for mussel-inspired crosslinking) or a coppercatalyzed click crosslinking, or both (dual crosslinking, Scheme 2).To resolve the foreseen mutual inhibition effect between PI(oxidant) and Cu(II) reductant (sodium L-ascorbate, NaLAc, toreduce Cu(II) into Cu(I)), a zwitterionic organic chemical bufferingagent, HEPES, was introduced into the system as it is widely used incell culture as a buffering agent. The two adjacent tertiary aminestructures in HEPES as found similarly in the chemical structures ofpreviously used copper-chelating agents, can chelate copper[19,30e35]. With the use of HEPES, the mutual inhibition effectbetween PI and NaLAc was successfully minimized and the cyto-toxicity caused by the copper catalyst was also significantlyreduced. The dual-crosslinked iC-X-PI bioadhesives showedsignificantly enhanced tensile (cohesive) and adhesive strength, aswell as prolonged degradation, making mussel-inspired adhesivepolymers well suited for many surgical adhesion applications. It isalso worthwhile to note that the iC-X-PI bioadhesives exhibitedintriguing antimicrobial properties due to the presence of inher-ently anti-microbial citrate and PI [12].

2. Experimental section

1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) andpentaerythritol tribromide were purchased from AK Scientific, Inc.(Union City, CA, USA). Propiolic acid was purchased from Carbo-synth Ltd. (Compton, UK). All other chemicals were purchased fromSigma-Aldrich and were used without further purification.

2.1. Characterization

Synthesized pre-polymers and their monomer componentswere dissolved in DMSO-d6 and CDCl3, respectively, and their 1HNMR spectra were recorded on 300 MHz Bruker DPX-300 FT-NMRspectrometer. The samples dissolved in acetone and prepared asthin cast films on KBr pellets were analyzed by Nicolet 6700 FTIRspectrometer for Fourier transform infrared (FTIR) spectra. UVevisspectra were also recorded on a UV-2450 spectrometer (Shimadzu,Japan).

2.2. Monomer and polymer syntheses

The synthesis of click chemistry enhanced iCs (clickable iC) in-volves three steps: synthesis of the 3N3 monomer, synthesis of the2PL monomer, and then synthesis of functionalized pre-polymersby polycondensation in a facile, one-pot reaction of citric acid(CA), poly(ethylene glycol) (PEG) diol, a catechol-containing com-pound such as dopamine or L-DOPA, along with either the 3N3monomer or the 2PL monomer to achieve the azide (iC-3N3) pre-polymer or the alkyne (iC-2PL) pre-polymer respectively.

2.2.1. Pentaerythritol triazide (3N3) monomer synthesisTo synthesize the 3N3 monomer, we modified the synthesis

process of 2, 2-Bis(azidomethyl)propane-1,3-diol (diazido-diolmonomer, DAzD), which contains similar azide group functionality[14,36e38]. Briefly, pentaerythritol tribromide (32.48 g, 0.10 mol)and sodium azide (NaN3) (26.87 g, 0.42mol) were heated at 120 �Covernight in a round-bottom flask with 120 mL of dime-thylformamide (DMF), stirring. Following, we removed the solventat 80e120 �C under vacuum, re-dissolved the crude product inacetone, filtered out the salt, and then removed acetone by rotaryevaporation. We then extracted our product by dissolving indichloromethane (DCM, 200 mL) and saturated saline (50 mL�3),so that the organic phase was separated and dried by anhydrousmagnesium sulfate (MgSO4). After filtration and rotatory

HO OH

OH

OH

O O

O+ HO

OHn

NH2HO

HO

PEGwithcitric acid (CA)

iC-3N3

O OO

O

HO

N3 N3N3

or

OH

Br Br

HO

Br

NaN3 N3 N3

HO

N3

OH

HO OH+ OH

OOH

O O

H2SO4toluene, reflux

OO

(A)

(B)

(C)

3N3 monomer

2PL monomer

pre-polymers

O

OO O

XOO

R1

R2m

R1 = H or polymer chain

R2 =

X = -O- or -NH-

OH

OH

N3

N3

N3

O

O

OO

or

or

n

R2 = OH

OHiC-2PL

Scheme 1. Synthesis schemes of triazide (3N3) (A) monomer, dipropiolate (2PL) (B) monomer, and clickable injectable citrate-based mussel-inspired bioadhesive pre-polymers (iC-3N3 and iC-2PL) (C).

Scheme 2. Dual crosslinking (oxidant and click (CuAAC)) of clickable iC (mixture of iC-3N3 and iC-2PL).

J. Guo et al. / Biomaterials 112 (2017) 275e286 277

evaporation, we obtained the final product (19.9 g, 94% yield). 1HNMR (Figure S1, 300 MHz; CDCl3; d, ppm) of 3N3 monomer: 3.38 (3,s, eCH2eN3), 3.54 (1, s, eCH2eOH). FTIR of 3N3 monomer (Fig. 1A,cast on KBr, cm�1): 2103 (strong, eN3).

2.2.2. Trimethylolethane dipropiolate (2PL) monomer synthesisWe adapted the synthesis of 2PL monomer from literature [39],

in which 1,1,1-tris(hydroxymethyl)propane (13.4 g, 0.1 mol) andpropiolic acid (14.01 g, 0.2 mol) was dissolved in 120 mL dry

toluene and placed in a dry 250 mL round-bottom flask equippedwith a Dean-Stark trap and a magnetic stir bar, adding four drops ofsulfuric acid (H2SO4) as catalyst and heating to 125 �C to reflux for24 h.We performed purification by removing toluene, re-dissolvingthe crude product in ethyl acetate (150 mL), washing with 5% so-dium bicarbonate (NaHCO3) solution (30 mL�2) and shortly afterwith a brine solution (30 mL�2). Thereafter, we dried our productover anhydrous MgSO4, filtered, and removed the remaining sol-vent by rotary evaporation, so that the final product was obtainedas slightly yellow oil (21.9 g, 92% yield). 1H NMR (Figure S1,300 MHz; CDCl3; d, ppm) of 2PL monomer: 0.91 (br, eCH2eCH3),1.48 (br, eCH2eCH3), 2.94 (s, eOOC^CH), 3.55 (br, eCH2eOH), 4.18(br, eCH2eOOC^CH). FTIR of 2PL monomer (Fig. 1B, cast film onKBr, cm�1): 2118 (strong, eOOC^CH), 1709 (eCH2eOOC^CH).

2.3. Clickable iCMBA pre-polymer synthesis

The general synthesis of iC pre-polymers is adapted from ourprevious work [6,11,12], to which we introduced clickable func-tionalities in this work. For example, iC-P4-3N3 denotes a pre-polymer, in which P4 refers to the molecular weight of PEG (PEG-400) and 0.2 indicates the feed ratio of 3N3 monomer to PEG usedin the formulation. Since the feeding ratio of dopamine to PEG usedin all of our formulations was 0.3, it was omitted in the formulanames for convenience. Briefly, we heated citric acid (CA) andPEG400 with a monomer ratio of 1.2:1 in a round-bottom flask at160 �C to obtain a clear mixture, into which we added dopamine(0.3 molar ratio to PEG) under N2 at a reduced temperature of140 �C. At a further reduced temperature of 120 �C, we next addedthe 3N3 monomer (0.2 molar ratio to PEG), reacting for ~36 h undervacuum and quenched with deionized (DI) water. The dissolvedpre-polymer was purified by dialysis against water (molecularweight cut-off (MWCO) of 1 kDa), and then freeze-dried to obtainthe purified pre-polymer iC-P400D0.3e3N3-2.

Fig. 1. FTIR spectra of iC-3N3 (A) and iC-2PL (B) pre-polymers; 1H NMR spectra of alkyne functionalized gelatin (Gelatin-Al, GL, C); and UVevis spectra (D) of iC-3N3 and iC-2PL pre-polymers.

J. Guo et al. / Biomaterials 112 (2017) 275e286278

FTIR of iC-P4-3N3 (Fig. 1A, by casting pre-polymer solution inacetone on KBr slice, cm�1): 2103 (eN3) and 1723 (COOe), 1634(CONHe). 1H NMR of iC-P4-3N3 (Fig. S1A, 300 MHz; DMSO-d6; d,ppm): 2.69e3.03 (m, eCH2e from CA), 3.30e3.78 (br, e(CH2)2eOefrom PEG and eCH2e from 3N3 monomer), 4.10, 4.16 (br, eOeCH2eCH2eOOCe from the terminals of PEG that connected to estergroups), 6.26, 6.49, 6.65, 8.83 (m, protons from dopamine).

The iC-P4-2PL (pre-polymer with alkyne groups) was synthe-sized likewise, using 2PL instead of 3N3 in equal molar ratios. FTIRof iC-P4-2PL (Fig. 1B, by casting pre-polymer solution in acetone onKBr slice, cm�1): 2123 (eOOC^CH) and 1730 (COOe), 1628 (CON-He). 1H NMR of iC-P4-2PL (Fig. S1B, 300 MHz; DMSO-d6; d, ppm):0.79 (br, eCH2CH3 from 2PL monomer), 2.65e2.96 (m,eCH2e fromCA, eOOC^CH from 2PL monomer), 3.36, 3.53 (br, e(CH2)2eOefrom PEG and eCH2e from 2PL), 4.10, 4.16 (br, eOeCH2eCH2e

OOCe from the terminals of PEG that connected to ester groups),6.26, 6.49, 6.65, 8.72. 8.85 (m, protons from dopamine).

In order to increase the crosslinking density of iCs, PEG400 andPEG400/PEG200 mixture (mol/mol ¼ 1/1) were also used in thispaper, and the compositions of the obtained pre-polymers, iC-P2/4-3N3 and iC-P2/4-2PL, are listed in Table 1. Normal iCMBA pre-

Table 1Pre-polymer identification, feeding ratios, and dopamine contents.

Pre-polymer name MW of PEG (Da)

iC-P4 400iC-P4-3N3 400iC-P4-2PL 400iC-P2/4-3N3

(PEG 200 and 400, mol/mol ¼ 1/1)200 and 400

iC-P2/4 -2PL 200 and 400

polymer iC-P4 was also synthesized as control.The successful incorporation of dopamine into iCMBA and

clickable iCMBA pre-polymers was also proven by 1H NMR (Fig. 1Aand B) and UVevis spectra (Fig. 1D), and the dopamine contents inthese pre-polymers were determined by UVevis spectra (Fig. 1Dand Table 1).

2.4. Alkyne functional gelatin (Gelatin-Al, GL) synthesis

To prepare GL as outlined in Scheme S1, we first synthesizedmonopropargyl succinate (AleCOOH) through a ring opening re-action of succinic anhydride (20 g, 0.2 mol) using propargyl alcohol(11.64 g, 0.2 mol) and a catalyst, 4-dimethylaminopyridine (DMAP)(0.98 g, 0.008 mol, 4mol% to alcohol) (Scheme S1A) in 120 mLacetone in a 250 mL round-bottom flask. After 8 h of refluxing at80 �C, acetone was removed under vacuum, and the crude productwas re-dissolved in EtAc (200 mL) and washed with brine(30 mL�3). The organic phase was dried over anhydrous MgSO4,filtered, and the solvent was removed by rotary evaporation. Afterconducting recrystallization in EtAc/hexane (v/v¼ 1/1) three times,the final product was obtained as a slightly yellow crystal (30.0 g,

Feeding ratio of CA: PEG: dopamine:3N3 or 2PL monomer

Dopamine content inpre-polymer (mmol/g)

1.1: 1: 0.3: 0 0.5701.2: 1: 0.3: 0.2 0.5541.2: 1: 0.3: 0.2 0.4521.2: 1: 0.3: 0.2 0.874

1.2: 1: 0.3: 0.2 0.547

J. Guo et al. / Biomaterials 112 (2017) 275e286 279

96% yield). 1H NMR (Fig. 1C, 300 MHz; CDCl3; d, ppm) of monop-ropargyl succinate: 2.51 (s, eC^CH), 2.66 (s, HOO-CeCH2eCH2eCOOe), 4.68 (s, eCH2eC^CH), 9.46 (br,HOOCeCH2e).

Next, gelatin-Al was synthesized by reacting AleCOOH andgelatin in dimethyl sulfoxide (DMSO) in the presence of N-hydroxysuccinimide (NHS) and EDC (Scheme S1B). This required aninitial activation of AleCOOH (1.56 g, 10 mmol) by NHS (1.16 g,10.1 mmol) and DMAP (0.122 g, 1 mmol) dissolved in 15 mL ofDMSO. Next, EDC (1.94 g, 10.1 mmol) was added at 0 �C; the reac-tion mixture was allowed to warm to room temperature and to bereacted for another 24 h. Then, gelatin (5 g, from bovine skin,Sigma) dissolved in 40 mL DMSO was added to the NHS activatedAleCOOH solution, and the reaction mixture was stirred foranother 24 h. After dialysis (MWCO: 1000Da) against DI water for 3days following by freeze-drying, GL was obtained as a pale yellowpowder (10.8 g, yield: 94%). The 1H NMR and FTIR spectra of GL andunmodified gelatin are shown in Fig. 1C and S2.

2.5. Crosslinking of clickable iCs and setting time measurement

Three different routes were conducted on the iC-3N3/2PL pre-polymer (equal-weight mixture of iC-3N3 and iC-2PL): oxidationof catechol groups by sodium (meta) periodate (PI); CuAAC (clickreaction); and dual crosslinking by PI and CuAAC.

2.5.1. iC-3N3/2PL mixture crosslinking by PI (iC-BD-PI, BD meansblending)

The crosslinking of iC-3N3/2PL equal-weight mixture by PIadapted from our previous work [6,11,12]. For 2 g of 50 wt% iC-3N3/2PL pre-polymer solution, 2 mL of 8 wt% PI solution was used. Thegel times of iC-P4-3N3/2PL mixture and iC-P2/4 -3N3/2PL mixturecross-linked by 8 wt% PI are listed in Table 2.

2.5.2. Crosslinking of iC-3N3/2PL mixture wtih CuAAC (iC-X)Since an iC-3N3/2PL mixture contains both azide and alkyne

groups, it can be crosslinked through CuAAC. The obtained cross-linked gel is named iC-X for convenience. CuSO4 and sodium L-ascorbate (NaLAc) catalyst system that can generate Cuþ ions in situwere used for the CuAAC. For example, for 2 g of 50 wt% iC-3N3/2PLpre-polymer solution, a 2 mL solution containing 5 mM CuSO4 and50 mM NaLAc was used as a copper catalyst. For varied concen-trations of CuSO4 and ratios of NaLAc/CuSO4, their correspondinggel timeswere recorded as listed in Fig. 2A. Considering that the useof oxidant, such as PI in the dual crosslinking (PI and click) process,will affect the reduction of Cu2þ to Cuþ by the NaLAc. To reduce theside effects of the oxidant to click reaction, HEPES, a biocompatiblebuffer agent often used in cell culture, was used as a copper ion

Table 2Gel times of different iCs and clickable iCs crosslinked by various concentrations of sodconcentration used was 50 wt%).

Pre-polymer namea PIb concentration (wt%) PI to pre-polymer ratio (wt%)

iC-P4 8 8iC-P4-3N3/2PL 8 8iC-P4-3N3/2PL 0 0iC-P4-3N3/2PL 0 0iC-P4-3N3/2PL 4 4iC-P4-3N3/2PL 4 4iC-P4-3N3/2PL 2 2iC-P4-3N3/2PL 2 2iC-P2/4-3N3/2PL 4 4

a For clickable pre-polymer mixtures, for example, iC-P4-3N3/2PL, the weight ratio beb PI: sodium periodate.c NaLAc: sodium L-ascorbate.d HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

chelating agent to protect and stabilize the reduced Cuþ ions. Theeffect of HEPES to the gel times was investigated as shown inFig. 2B. The optimized HEPES concentration was determined to be20mM,which generated the fastest crosslinking in our investigatedformulations for only using CuSO4-NaLAc.

2.5.3. Dual crosslinking of iC-3N3/2PL mixture with PI and CuAAC(iC-X-PI)

The dual crosslinking of iC-3N3/2PL mixture was conducted atthe presence of both copper catalyst and PI. For example, 2 g of50 wt% iC-3N3/2PL pre-polymer solution, click catalyst and oxidantsolution with a total volume of 2 mL were added separately andcombined in the final solution with 5 mM CuSO4, 50 mM NaLAc,20 mM HEPES, and 4 wt% PI. The effect of HEPES to the gel times ofiC-X-PI was investigated at room temperature (Fig. 2C). The geltimes of iC-X-PIs cross-linked at 37 �C are shown in Fig. 2D.

2.6. Rheological tests

Rheological tests were undertaken by using an AR-G2 rheom-eter (TA Instruments, UK) in a parallel plate configuration, byemploying sandblasted stainless steel 40 mm diameter platesthroughout and by using a Peltier plate for temperature control. In atypical rheological test for gelling kinetics, 800 mL of iC-P4-BD(equal-weight blend 50 wt% solution in DI water of iC-P4-3N3 andiC-P4-2PL) was mixed with 800 mL 8 wt% PI solution in DI water (orcopper catalyst, or the mixture of copper catalyst and PI, the finalvolumewas 800 mL) and themixturewas applied to the lower plate,which was preheated to 25 �C. The upper plate was immediatelybrought down to a plate separation of 0.5 mm and the measure-ment was taken. A low frequency of 1 Hz and 1% strain was appliedto minimize interference with the gelation process and to keep themeasurement within the linear viscoelastic region. The gelationkinetics was measured in a time sweep by the monitoring of thechange of storage (G0) and loss (G00) moduli as a function of time. Allmeasurements were repeated in triplicates.

2.7. Properties of cross-linked clickable iCs

To investigate the properties of clickable iCs crosslinked by PI,CuAAC, or both, we evaluated mechanical properties according toASTM D412A (Instron, Norwood, MA) [6,11,12]. The iC samples atboth dry and wet states (swollen in water for 4 h) were cut intostrips (25 mm � 6 mm � 1.5 mm, length � width � thickness),placed in the mechanical tester, and pulled to failure at a rate of500 mm/min. The adhesion strengths of different clickable iC for-mulations were measured using the lap shear strength test ac-cording to a modified ASTM D1002-05 [6,11,12]. Briefly, porcine-

ium periodate (PI) or copper catalyst, or both at room temperature (pre-polymer

CuSO4 (mM) NaLAcc (mM) HEPESd (mM) Measured gel time (s)

0 0 0 163 ± 90 0 0 155 ± 85 50 0 142 ± 65 50 20 61 ± 55 50 0 663 ± 65 50 20 479 ± 75 50 0 529 ± 45 50 20 359 ± 65 50 20 368 ± 5

tween iC-P4-3N3 and iC-P4-2PL was 1/1.

0 5 10 150

120

240

360

480

600

720

840

960

1080

1200

CuSO4 concentration (mM)

Gel

tim

e (s

)

CuSO /NaLAc = 1/2CuSO /NaLAc = 1/5CuSO /NaLAc = 1/10

A

0 10 20 300

60

120

180

240

300

360

420

480CuSO /NaLAc = 1/2CuSO /NaLAc = 1/5CuSO /NaLAc = 1/10

Gel

tim

e (s

)

HEPES concentration (mM)

[CuSO ] = 5 mM

B

0 2 4 6 80

60

120

180

240

300

HEPES: 20 mMG

el ti

me(

s)

PI concentration(wt%)

Cu ; NaLAc: 5; 50 mMCu ; NaLAc: 10; 50 mM

37 C

D

0

10240

300

360

420

480

540

600

660

Gel

tim

e (s

)

Cu : 5 mM, NaLAc: 50 mM, PI 2wt%Cu : 5 mM, NaLAc: 50 mM, PI 4wt%Cu : 10 mM, NaLAc: 50 mM, PI 2wt%Cu : 10 mM, NaLAc: 50 mM, PI 4wt%

20 mM HEPESno HEPES

C

Fig. 2. Gel times of iC-P4-3N3/2PL equal-weight mixture crosslinked by different formulations of CuSO4-sodium L-ascorbate (NaLAc) (A); the effect of HEPES to the gel times of iC-P4-3N3/2PL equal-weight mixture crosslinked by CuSO4-NaLAc (B) and dual-crosslinked by CuSO4-NaLAc and PI (C); gel times of dual-crosslinked iC-X-PI at 37 �C (D, X ¼ click).

J. Guo et al. / Biomaterials 112 (2017) 275e286280

derived, acellular small intestine submucosa (SIS) material strips(40 � 4 mm) (OASIS®, HealthPoint Ltd. TX) were adhered to theterminals of albumin strips (40 � 100 mm) using superglue (3 MScotch). The clickable iC formulations (20 mL in total volume) werein-situ mixed and applied onto the two SIS terminals of two albu-min strips, which were pre-soaked in PBS (pH 7.4) to mimic the wetenvironment of clinical settings. The two strips were pressedagainst each other to form a bond and the attached strips wereplaced in a highly humid chamber for 2 h. The lap shear strength ofbonded strip specimens was subsequently measured using Instronmechanical tester (Norwood, MA) fitted with a 10 N load cell at acrosshead speed of 1.3 mm/min. Detailed methods for evaluatingsol contents, swelling ratios, and degradation profiles have beendescribed previously [6,11,12]. Degradation studywas conducted byincubating dried crosslinked samples in PBS (pH 7.4) at 37 �C. Massloss was recorded at preset time points.

2.8. Cyto-compatibility evaluations of clickable iC pre-polymersand cross-linked clickable iC hydrogels

The biocompatibility evaluations of clickable iC pre-polymers,GL, and cross-linked clickable iC hydrogels were conductedsimilar to our previous work using Human-derived mesenchymalstem cells (hMSCs, ATCC® PCS-500-012TM) as cell model. Cells atpassages 5e10 were used for cytotoxicity study in this work.[6,11,12], in which sol contents and degradation products of click-able iC hydrogel, crosslinked by PI, CuAAC, or both were evaluatedfor cytotoxicity by respectively setting iC-P4 PI 8 wt% or commer-cially available PLGA (LA/GA ¼ 50/50, Mw~60KDa, purchased fromPolyscitech) as control [11]. To evaluate the cytotoxicity of pre-polymers, 10, 1, and 0.1 mg/mL pre-polymer solutions were pre-pared by directly dissolving pre-polymers in complete Dulbecco'smodified eagle's medium (DMEM, growth media, MG). Poly(-ethylene glycol) diacrylate (molecular weight ~700Da) was used as

control. The sol content solutions or degradation products ofdifferent formulations were obtained by incubating equal mass(0.5 g) hydrogel specimens in 5 mLs of PBS (pH 7.4, for sol content)at 37 �C for 24 h (sol) or in 5mLs of 0.1 M NaOH solution (fordegradation product) till full degradation (degradation), respec-tively. Subsequently, three different dilutions of degraded productsor leachable parts were prepared: 1�, 10� and 100� (1� was thesolution of degradation products or leached parts with no dilution;10� and 100� indicate 10-fold and 100-fold dilutions of 1� solu-tion diluted with PBS, respectively). 200 mL of hMSC suspension inMG medium (5�104 cells/mL) was dispensed in each well of a 96-well plate and incubated for 24 h. Then, 20 mL of sol content/degradation product solutions at various concentrations wereadded into the culture media in the plate and the cells were incu-bated for another 24 h before their numbers were quantified with aMTT assay. All the solutions above were adjusted to neutral pH andfiltered with sterile 0.2 mm filters prior to use for cell culture. Wealso studied cell attachment and proliferation of hMSC cells oncrosslinked iC-P2/4-X-PI 4 wt% films using the process describedpreviously [6,11,12].

2.9. Antimicrobial performance of cross-linked clickable iChydrogels

The antimicrobial performance, including short-term and long-run antibacterial and antifungal performance, of crosslinked click-able iC hydrogels was conducted using Staphylococcus aureus(S. aureus) and Escherichia coli (E. coli) as respective positive andnegative bacteria models, and by using Candida albicans(C. albicans) as a fungi model.

2.9.1. Bacterial incubationStaphylococcus aureus (S. aureus, ATCC® 6538™) and Escherichia

coli (E. coli, ATCC® 25922™) were purchased from ATCC (American

J. Guo et al. / Biomaterials 112 (2017) 275e286 281

Type Culture Collection) and used per safety protocols. Tryptic soyagar (Cat. #: C7121) and tryptic soy broth (Cat. #: C7141) forculturing S. aureus were purchased from Criterion. Luria broth base(LB broth, Cat. #: 12795-027) and select agar (Cat. #: 30391-023)for culturing E. coli were purchased from Invitrogen. S. aureus andE. coli were cultured at 37 �C in sterilized tryptic soy broth and LBbroth, respectively with a shaking speed of 150 rpm in a rotaryshaker for 24 h and the obtained bacteria suspensions were dilutedinto desired concentrations before use.

2.9.1.1. Anti-bacterial performance of crosslinked clickable iC hydro-gels. We measured the kinetics of bacterial inhibition according toour method described previously [12]. Briefly, S. aureus and E. coliwere cultured onto crosslinked clickable iC hydrogels using iC-P2/4-BD-PI 4 wt%, iC-P2/4-X, and iC-P2/4-X-PI 4 wt% as the representativeexperimental groups. As controls, we used iC-P4-PI 4 wt% andPEGDA/2-Hydroxyethyl methacrylate (HEMA) hydrogels (1:1 w/wfor PEGDA to HEMA, with PEGDA ~ 700 Da) [40,41], as well ascommercially available Hydrofera Blue bacteriostatic foam dressing[42]. Briefly, 0.2 g of test sample was immersed in 20 mL of a germ-containing broth solution with a bacterial concentration of 100�diluted from optical density (OD) at 600 nm around 0.07. Themixture was then incubated at 37 �C with shaking speed of150 rpm. 200 mL of the mixture was taken out at each pre-set time-point, and the OD value of themedium at 600 nmwas recorded by amicroreader (TECAN, infinite M200 PRO). We calculated inhibitionratios of hydrogels or Hydrofera Blue according to equation (1):

Inhibition ratioð%Þ ¼ 100� 100� At � Ao

Acon � Ao(1)

where Ao is the starting optical density (bacteria in media prior toincubation), which is incubated at 37 �C for the predetermined timepoints to obtain Acon (pure medium control) or At (medium con-taining hydrogel).

2.9.1.2. Long-term anti-bacterial evaluation of crosslinked clickableiC hydrogels. We also simulated long-term antibacterial capabilityof our hydrogels by evaluating degradation products and periodicalrelease solutions against S. au and E. coli based on the bacterialinhibition ratio test, described previously [12].

2.9.2. Anti-fungal studyFungi (Candida albicans, C. albicans) purchased from ATCC

(ATCC® 10231™) were used per safety protocols. Yeast malt (YM)medium broth (Lot #: 1964C030) and agar (Lot #: 1964C030) forfungi culture were purchased from Amresco and Acumedia,respectively. Tween 20 added to the YM broth medium at a finalconcentration of 0.5 wt% was sterilized before use. C. albicans wasmaintained on YM agar plates in all cases. For experiments,C. albicans cells were scraped from YM agar plates and dispersed ina Tween 20 (0.5 wt%)-containing YM broth. The cells were countedwith a hemocytometer and were diluted to obtain a final fungiconcentration of 0.5e1�107 cells/mL [12]. The measurement of theexact amount of fungi used a colony growth assay on YM agarplates, which will be described in detail below.

2.9.2.1. Anti-fungal study performance of cross-linked clickable iChydrogels. Following incubation of C. albicans [12,43], we evaluateddirect exposure antifungal effects of cross-linked clickable iChydrogels based on iC-P2/4-BD-PI 4 wt%, iC-P2/4-X, and iC-P2/4-X-PI4 wt% as the representative experimental groups and iC-P4-PI 4 wt%, PEGDA/HEMA hydrogels and Hydrofera Blue as controls [12].

2.9.2.2. Long-term anti-fungal evaluation of crosslinked clickable iChydrogels. We also simulated long-term antifungal capability of ourhydrogels by evaluating degradation products and periodicalrelease solutions against C. albicans based on the bacterial inhibi-tion ratio test, described previously [12].

3. Results and discussion

3.1. Synthesis and characterization of clickable iC pre-polymers andGL

Challenges to mussel-inspired polymers include fast oxidationof catechol groups on L-DOPA or its derivatives (i.e. dopamine) if leftunprotected under neutral or basic conditions [2,44,45]. Thus ourapproach based on iCs conveniently provides L-DOPA or dopaminewith a slightly acidic environment mainly imparted by citric acid,enabling facile and low-cost one-pot polycondensation reactionswithout further protection of these groups [11,12]. At the sametime, our iC platform introduces versatile clickable functionalitiesby the same polycondensation method. In order to maintain theelasticity of modified iCs and to take into consideration the largereactivity difference between long-chain PEG and short-chainazide/alkyne functionalized diols used in POC-click development[14], azide- or alkyne-functionalized monohydroxyl compounds(Scheme 1A, B) were introduced concurrently with dopamine, asfunctional monomers, into the side chains of iCs to form azide- oralkyne-functionalized iC, iC-3N3 or iC-2PL pre-polymers, respec-tively (Scheme 1C).

The FTIR spectra of representative iC-3N3 and iC-2PL pre-poly-mers, iC pre-polymers, and corresponding 3N3 and 2PL monomersare shown in Fig.1A and B, respectively. The successful introductionof 3N3 monomers into the iC-3N3 pre-polymer was confirmed bythe characteristic infrared absorption peak of azide at 2100 cm�1,whichmatches the azide peak of 3N3 monomers (Fig. 1A). Likewise,the appearance of characteristic infrared peak of alkyne (around2123 cm�1, Fig. 1B) and the 1H NMR peak of the protons eCH2CH3(around 0.93 ppm, Fig. S1 right) from 2PL monomer confirmed theincorporation of alkyne groups into iC-2PL. Similar multiple peaksat 6.4e6.7 ppm in the 1H NMR spectra of both clickable iCs andnormal iC were assigned to the protons of phenyl group fromdopamine [11,12], indicating that click functionalization of iC didnot affect the function of catechol groups (Fig. S1). The dopaminecontents of clickable iCs and iC pre-polymers were determined bymeasuring the UVeVis absorbance at 280 nm, then comparing tothe dopamine standard curve (Table 1). To maintain the molar ra-tios of dopamine: PEG at 0.2: 1, the amount of dopamine wasreduced accordingly for the iCs made from PEG with high molec-ular weights (Fig. 1D and Table 1). The UVeVis absorbance spectraof all clickable iC and iC pre-polymers are shown in Fig. 1D. GL wasalso synthesized as illustrated in Scheme S1. The modification ofgelatin with alkyne groups was confirmed by proton peaks fromeCH2eC^CH in the 1H NMR spectrum of GL (a in Fig. 1C). Therewas no significant difference between the FTIR spectra of GL andgelatin (Fig. S2).

3.2. Crosslinking of clickable iC pre-polymers by CuAAC, PI, or both

Possessing both catechol groups and click functionalities, themixture of iC-3N3 and iC-2PL pre-polymers can undergo mussel-inspired intermolecular crosslinking via oxidation of catecholgroups by the use of an oxidant (such as PI) as shown in our pre-vious work [11,12], or CuAAC, or both. For the CuAAC, CuSO4-NaLAccatalyst system is the most commonly used catalyst for the aqueoussystem. It can form Cu(I) in situ upon mixing CuSO4 (a stable Cu(II)salt) and NaLAc (a reductant) [23,25,29]. First, we assessed the

Fig. 3. Rheological analysis on the gelation of iC-P4-3N3/2PL (1:1 weight ratio) solution(50 wt%) crosslinked by oxidant (8 wt% PI, black color), CuAAC (CuSO4-NaLAc-HEPES,5-50-20 mM, blue color), or dual-crosslinking (by CuSO4-NaLAc -HEPES (5-50-20 mM)and 4 wt% PI together, red color) at 25 �C. (For interpretation of the references to colourin this figure legend, the reader is referred to the web version of this article.)

J. Guo et al. / Biomaterials 112 (2017) 275e286282

cytotoxicity of CuSO4, NaLAc and CuSO4eNaLAc systems (CuSO4:NaLAc ¼ 1:10, 1:5, or 1:2) by using the MTT (methylthiazolyl-diphenyl-tetrazolium bromide) assay against human-derivedmesenchymal stem cells (hMSCs) (Fig. S3). As shown in Fig. S3,both Cu2þ and NaLAc treated hMSCs showed cell viability higherthan 85% when the concentrations were lower than 1000 mmol/L.For the CuSO4-NaLAc system, cytotoxicity was also within theacceptable range when the concentrations of copper ion werelower than 1000 mmol/L, especially at the molar ratios of CuSO4 toNaLAc of 1:5 and 1:2. For the equal-weight mixture of iC-3N3 andiC-2PL (clickable iC) crosslinked by CuAAC using different concen-trations of CuSO4 and different CuSO4 to NaLAc ratios, the gel timesare shown in Fig. 2A. Higher CuSO4 concentrations and lowerCuSO4/NaLAc ratios led to faster crosslinking, even below a minutefor certain formulations.

Both CuSO4-NaLAc and PI were used to enable dual crosslinkingof the clickable iC system. When both CuSO4-NaLAc and PI wereused in the system, the gel time, determined by a vial tilting test,wasmuch longer than that of the same system crosslinked by eitherCuAAC or PI [11] (Fig. 2C). This phenomenon suggests that thereshould be a mutual inhibition effect between PI (oxidant) andNaLAc (reductant). We thus hypothesized that a copper chelatingagent that can protect reduced Cu(I) ions from the oxidant (PI) usedin the system may resolve this problem. A zwitterionic organicchemical buffering agent, HEPES, was selected, mainly because it iswidely used in cell culture and contains two adjacent tertiaryamine structures that are also found in other copper-chelatingagents [19,30e35]. Our hypothesis was confirmed by the gel timetest (Fig. 2C). After the addition of HEPES, the gel times of clickableiC systems crosslinked by CuSO4-NaLAc and PI combined werereduced (Fig. 2C). The effect of HEPES on the gel time of the samesystem crosslinked solely by CuAAC was also studied. Among theinvestigated formulations, 20 mM of HEPES was found to generatethe quickest crosslinking for all the three CuSO4/NaLAc ratios used(Fig. 2B). Based on this, we determined the optimized concentra-tion of HEPES for crosslinking to be 20 mM, a relatively safe dosagefound to be cytocompatible with hMSCs (Fig. S3) and typicallybelow dosages used in some DMEM formulations. The gel times ofdual-crosslinked clickable iCs crosslinked by CuSO4-NaLAc-PI-HEPES at 37 �C were below 5 min for all the formulations tested(Fig. 2D). When the concentrations of HEPES and CuSO4-NaLAcwere fixed, increasing the concentration of PI within the range of0e4 wt% led to prolonged gelation times (Fig. 2D). Gel time slightlydecreased when the concentration of PI was increased to 8 wt%.This decrease might be due to the relatively increased catecholgroup-based crosslinking over the click crosslinking (Fig. 2D). Therepresentative gel times of 1) iCs or clickable iCs, 2) crosslinked byeither PI or CuAAC, or both, and 3) with or without HEPES, are listedin Table 2.

3.3. Rheological characterization

The viscoelastic profiles of representative clickable iCs cross-linked by PI, CuAAC or both, including clickable iCs crosslinked by8 wt% PI, or CuSO4-NaLAc-HEPES (5-50-20 mM), or CuSO4-NaLAc-HEPES (5-50-20 mM) and 4 wt% PI, were evaluated by rheologicalstudies (Fig. 3). The gel times, as characterized by the crossoverpoint of the rheological storage (G0) and loss (G00) moduli, were inagreement with the gel times observed by vial tilting tests (Fig. 2Dand Table 2).

3.4. Properties of crosslinked clickable iCs

Next, we tabulated the mechanical properties of PI, CuAAC, ordual-crosslinked clickable iCs in both dry and fully hydrated states

(Fig. 4A, S4, Table S1). The dry mechanical properties (Fig. 4A andS4) reveal that the click (CuAAC) crosslinked iC films (iC-P4-X, iC-P2/4-X, and GL-iC-P2/4-X) exhibited comparable tensile strength andlower moduli, but possessed higher elongation at break comparedto those of the corresponding iC films crosslinked by PI (iC-P4-PI8 wt%, iC-P2/4-PI 8 wt%, and GL-iC-P2/4-PI 8 wt%). Contrarily, dual-crosslinked (with PI and CuAAC) iC films (iC-P4-X-PI 4 wt%, iC-P2/4-X-PI 4 wt%, and GL-iC-P2/4-X-PI 4 wt%) possessed both muchhigher tensile strength and moduli than the corresponding filmscrosslinked solely by either PI or CuAAC. The material with a higherelongation at break is more elastic and it can better resemble theelastic nature of soft tissues and withstand large amplitude andfrequency of relative movement between adhered tissue surfaces,which all render the material suitable for bioadhesion applications.It is worthwhile to note that the PI concentration used for dual-crosslinked films was 4 wt%, which is lower than the concentra-tion used for crosslinking films with only PI (8 wt%). It clearlydemonstrates the improvement of mechanical strength achievedby introducing click reactions to iCs. The addition of GL furtherimproved the mechanical strength (particularly moduli) of dual-crosslinked clickable iCs (Fig. 4A and S4). The stress-strain curvesof PI, CuAAC or dual-crosslinked clickable iCs are shown in Fig. 4A.All of the crosslinked iCs exhibited elastic behavior with theirelongation at break higher than 150%. CuAAC crosslinked clickableiCs possessed the highest elongation at break as some of them hadelongation at break higher than 500% (Fig. 4A and S4). The me-chanical strength of iCs decreased after they were hydrated andswollen and it is due to their hydrophilicity derived from hydro-philic PEG (Table S1).

The degradation studies of crosslinked clickable iCs demon-strate that the rate of degradation was indeed inversely propor-tional to the degree of crosslinking, and that the inclusion of gelatindid not prolong the degradation time (Fig. 4B). Dual-crosslinkedclickable iCs possessed prolonged degradation times as comparedto corresponding clickable iCs crosslinked solely by either PI orCuAAC. Complete degradation could be realized in 35 days in allformulas tested. It is a remarkable feature as the degradation pro-files of dual-crosslinked clickable iCs are well suited for manysurgical adhesive applications.

4-BD-PI 8wt%

Sol c

onte

nt (%

)

iC-P -PI 8wt%

BD=blend;X=click;GL=gelatin-alkyne

**

C

0

500

1000

1500

2000

2500

3000

3500 BD=blend;X=click;GL=gelatin-alkyne

Swel

ling

ratio

(%)

GL-iC-P-X

GL-iC-P-X-PI 4wt%

GL-iC-P-BD-PI 8wt%

iC-P-X-PI 4wt%

iC-P-X

iC-P-BD-PI 8wt%

iC-P -X-PI 4wt%iC-P -X

iC-P -BD-PI 8wt%

iC-P -PI 8wt%

****

****

**

*

D

Fig. 4. Evaluation of mechanical properties, showing stress-strain curves (A), degradation profiles (B), sol contents (leachable fractions) (C), and swelling ratios (D) of clickable iCmixture crosslinked by either sodium periodate (PI) or copper-catalyst, or both.

20 40 60 80 100 120 140 160 180 200 220 240

FibrinGlue

GL-iC-P-X-PI 8

GL-iC-P-X-PI 4

GL-iC-P-X-PI 2

GL-iC-P-X

GL-iC-P-BD-PI 8

iC-P -X-PI 8wt%

iC-P -X-PI 4wt%

iC-P -XiC-P -X-PI 2wt%

iC-P -BD-PI 8wt%

Lap Shear Strength (kPa)iC-P -PI 8wt%

J. Guo et al. / Biomaterials 112 (2017) 275e286 283

Sol contents and swelling ratios of different clickable iCs areshown in Fig. 4C and D. Although CuAAC enabled fast crosslinkingof clickable iCs in less than 2 min (Figs. 2 and 3), both the solcontents and swelling ratios of hydrogels crosslinked only byCuAAC were much higher than those of the corresponding hydro-gels crosslinked either by PI or dual crosslinking mechanism. Itindicates that click crosslinked clickable iCs (iC-X) possessed lowcrosslinking densities, whereas dual-crosslinked clickable iCs (iC-X-PI) possessed much lower sol contents (all < 5%) than iCs cross-linked by PI (~17.5%) (Fig. 4C). The swelling ratios of iC-X-PI werealso lower than those of normal iCs crosslinked by PI (Fig. 4D). Morependent carboxyl groups on iC's side groups consumed by hydro-phobic clickable monohydroxyl compounds are also considered tocontribute to the low sol contents and low swelling ratios of dual-crosslinked clickable iCs.

Fig. 5. Adhesion strength of fibrin glue, and normal iC and clickable iCs crosslinked bysodium periodate (PI), CuSO4-NaLAc-HEPES or both, onto wet porcine small intestinesubmucosa, determined by lap shear strength test.

3.5. Adhesion strength

The various formulations of clickable iC showed wet tissue lapshear strengths ranging from 41.21 ± 4.48 kPa (for iC-P4-X) to223.11 ± 15.94 kPa (for GL-iC-P2/4-X-PI 4 wt%) (Fig. 5). The intro-duction of click chemistry and the application of dual crosslinkingmechanism vastly improved the wet adhesion strength of iCs (from~50 kPa for iC-P4-PI 8 wt% to ~223 kPa for GL-iC-P2/4-X-PI 4 wt%).The inclusion of gelatin also helped increase the adhesion strengthof iCs as shown in Fig. 5. Most importantly, the highest adhesionstrength achieved (223.11 ± 15.94 kPa) during the test wasapproximately 13 times higher than that of commercially availablefibrin at 15.4 ± 2.8 kPa, which is widely considered the gold stan-dard [11].

3.6. In vitro evaluations of cytotoxicity and proliferation

The cytocompatibility of 1) clickable iC pre-polymers and GL, 2)sol contents and 3) degradation products of different crosslinkedclickable iCs were evaluated using hMSCs. In vitro cytotoxicity ofclickable iC pre-polymers tested was comparable to those of thecontrols, commercially available PEGDA (700 Da) and gelatin (fromcold fish skin) (Fig. 6A). The sol contents and degradation productsof different crosslinked clickable iCs formulations tested did notinduce any significant cytotoxicity against hMSCs (Fig. 6B and C).The above results, supported by qualitative cell morphology

Fig. 6. Cytotoxicity evaluation of clickable iC family: MTT assay for hMSCs cultured with: iC-click pre-polymers and Gelatin-Al (GL) (A), leachable part (sol content) (B) anddegradation product (C) of clickable iCs crosslinked through different routes for 24 h. Live/Dead assay for hMSCs seeded on dual-crosslinked iC-X-PI casted glass slides 1, 4, and 7days post cell seeding (D).

J. Guo et al. / Biomaterials 112 (2017) 275e286284

(Fig. 6D) suggested that clickable iCs and their different crosslinkedbioadhesive formulations are indeed cytocompatible.

3.7. Anti-bacterial performance of crosslinked clickable iCs

The antibacterial performance of clickable iCs crosslinked byeither PI, CuAAC, or both was assessed against representativeGram-positive and Gram-negative bacteria, S. au than in E. coli,using commercially available Hydrofera Blue bacteriostatic foamdressing and PEGDA/HEMA hydrogels as controls. The bacterialinhibition ratios tested by exposing bacteria-containing brothsdirectly to different crosslinked clickable iCs and other samples areshown in Fig. 7A and B. Inhibitionwas greater in S. au than in E. coli

for all tested normal and clickable iC formulations. Except the iC-Xhydrogels, all other formulations tested, including iC-P4-PI 4 wt%,iC-P2/4-BD-PI 4 wt%, and iC-P2/4-X-PI 4 wt%, exhibited comparableor even better bacterial inhibition effect than Hydrofera Blueagainst either S. aureus or E. coli, during the bacterial incubationperiods of 24 h. The bacterial inhibition ratios of iCs and clickableiCs crosslinked by PI against S. au or E. coli were still higher than90% or 60%, respectively, after 24 h. Even though the bacterial in-hibition ratios of iC-X-PIs dropped from ~100% after 13 h of incu-bation against S. aureus or after 8 h of incubation against E. coli, theyremained at ~70% against S. au or ~50% against E. coli even after 24 hof incubation. On the other hand, nearly no bacterial inhibitioneffect of PEGDA/HEMA hydrogels was observed.

Fig. 7. Antibacterial and antifungal performance of clickable iCs (iC-X) hydrogels: Inhibition ratios kinetics curves of crosslinked iC-X series, iC-P4-PI 4 wt%, PEGDA/HEMA (w/w ¼ 1/1, as negative control), and Hydrofera Blue (as positive control) against S. aureus (A) and E. coli. (B); Fungal survival ratios after direct exposure to crosslinked iC-click series, iC-P4-PI4 wt%, PEGDA/HEMA, and Hydrofera Blue for 3 h (C) (**p < 0.01). Long-term antimicrobial performance of iC-X series: Antifungal and antibacterial performance of degradationproducts at different dilutions (1�, 10�, and 100�) (D) and periodical release solutions (E) of crosslinked hydrogels: iC-X series, iC-P4-PI 4 wt%, PEGDA/HEMA (w/w ¼ 1/1, asnegative control), and Hydrofera Blue (as positive control).

J. Guo et al. / Biomaterials 112 (2017) 275e286 285

3.8. Anti-fungal performance of crosslinked clickable iCs

The antifungal performance was evaluated on the iC-X seriesand normal iCs by exposing them to a broth containing Candidaalbicans (C. albicans), fungi commonly seen in diabetic foot in-fections. Hydrofera Blue and PEGDA/HEMA hydrogels served con-trols, respectively. Based on the results shown in Fig. 7C, it can beseen that crosslinked iCs, including the CuAAC crosslinked one, allexhibited better fungal inhibition effect than Hydrofera Blue (~25%fungal survival). Especially, iCs crosslinked only by PI, had nearly nofungi grown after 3-h exposure. Although a bit weaker, dual-crosslinked clickable iCs still exhibited considerable fungal inhibi-tion: only ~10% fungal survival was observed. PEGDA/HEMAhydrogels also did not show any obvious fungal inhibition effect.

3.9. Long-term evaluations of antibacterial and antifungalproperties in crosslinked clickable iCs

Lastly, we evaluated long-term antibacterial and antifungalproperties of our hydrogels by incubating bacteria or fungi in 1x,10x, and 100x dilutions of degradation products or 4, 8, and 12-dayrelease solutions of crosslinked clickable iCs. Hydrofera Blue andPEGDA/HEMA hydrogels were set as controls. Survival ratios,measured after 24 h for bacteria and after 6 h for fungi, showed thatiC and clickable iCs, crosslinked by either PI or CuAAC, or both, all

exhibited great long-term inhibition, particularly against the Gram-positive S. au and fungi. Compared to our crosslinked iCs andclickable iC hydrogels, the Hydrofera Blue foam demonstratedweaker long-term antibacterial and antifungal properties becausethe small molecular antimicrobial agents (i.e. crystal violet andmethylene blue) that are simply mixed in the foam tend to bereleased rapidly in the early days of incubation in the broths [46].Due to the antimicrobial property of citric acid that was used tosynthesize iCs and clickable iC pre-polymers, crosslinked iCs andclickable iC hydrogels still possessed certain antimicrobial proper-ties even on day 8 and 14 after small molecular PI were mostlyreleased from the hydrogels [42].

4. Conclusions

In conclusion, copper-catalyzed azide-alkyne cycloaddition(CuAAC, click chemistry) was successfully introduced into inject-able citrate-based mussel-inspired bioadhesives (iCs). The biggestchallenge in this system, the mutual inhibition effect between so-dium (meta) periodate (PI, as oxidant used for catechol groupcrosslinking) and the Cu(II) reductant sodium L-ascorbate, (used toreduce Cu(II) into Cu(I) to form CuAAC), was successfully mini-mized by utilizing a biocompatible buffering agent often used incell culture, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), as a copper chelating agent. The introduction of click

J. Guo et al. / Biomaterials 112 (2017) 275e286286

chemistry into iCs as a secondary crosslinking mechanism greatlyimproved the cohesive (tensile) strength of iCs. Furthermore, theapplication of click chemistry freed additional catechol groups thatmay contribute to adhesion in a greater extent instead of mainlyparticipating in polymer crosslinking, leading to the design ofbioadhesives with vastly improved wet adhesion strength to bio-surfaces. Dual-crosslinked (PI and CuAAC) clickable iCs presentedintriguing antibacterial and antifungal abilities, which is beneficialfor the application of bioadhesives in hemostasis and wound/tissueclosure applications. Partial click functionalities were preservedeven after crosslinking, imparting our polymers with further po-tential to conjugate bioactive molecules and growth factors, as theconjugation of collagen mimetic peptide p15 in our previous paper[14], further improving the biocompatibility and bioactivity of iCs.

Acknowledgements

This work was supported in part by a National Cancer InstituteAward CA182670, and a National Heart, Lung, and Blood InstituteAward HL118498.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2016.10.010.

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